The invention generally relates to compositions and methods for identifying apoptosis signaling pathway inhibitors and activators.
Most cells in the body die through a non-inflammatory, energy-dependent form of cell death called apoptosis. Recent research into the molecular mechanisms of apoptosis has revealed that apoptosis is a genetically programmed process that can become deranged when the components of the cellular apoptotic machinery are mutated or present in inappropriate quantities. Dysregulation of apoptosis is associated with the pathogenesis of a wide array of diseases; cancer, neurodegeneration, autoimmunity, heart disease, and other disorders. Some diseases associated with increased apoptosis include AIDS, neurodegenerative disorders (e.g., Alzheimer""s disease, Parkinson""s disease, amyotrophic lateral sclerosis, retinitis pigmentosa), aplastic anemia, ischemic injury (e.g., myocardial infarction, stroke, reperfusion injury), and toxin-induced (e.g., alcohol induced) liver disease. Thus, apoptosis is critical not only during development and tissue homeostasis, but also in the pathogenesis of a variety of diseases. Products of genes involved in the regulation and execution of apoptosis are therefore potentially excellent targets for diagnosis and therapeutic intervention in disease processes, and they offer renewed hope for cures and treatments for a wide array of maladies [See Review by Rudin and Thompson, Ann. Rev. Med. 48:267-81 (1997)].
To date, various signaling pathways in apoptosis have been elucidated. Significant progress in defining the extracellular signaling mechanisms regulating apoptosis has been made in lymphocytes. B and T cells both express a wide variety of cell surface receptors that can either induce or inhibit apoptosis. The largest related group of these receptors, with at least 12 members, is the tumor necrosis factor (TNF) receptor (TNFR) family. These are characterized by cysteine-rich pseudorepeats in the N-terminal extracellular domains, through which each member binds to one or more of a family of TNF-related proteins. Under various experimental conditions, several of these receptors (including TNFR2, CD40, and CD30) inhibit apoptosis; others (including TNFR1 and Fas) induce apoptosis in lymphocytes.
The death-inducing receptors TNFR1 and Fas share a related intracellular sequence known as the death domain. FADD (Fas associated death domain protein), RIP, and TRADD (TNFR-associated death domain protein), can heterodimerize with the death domains of these receptors. FADD and RIP interact directly with Fas, whereas TRADD appears to bind only TNFR1. Over expression of any of these cytoplasmic proteins induces apoptosis. The TNFR1-TRADD complex forms only when TNFR1 has engaged extracellular TNF, and the complex was recently shown to secondarily bind FADD by heterodimerization of the C-terminal death domains of TRADD and FADD. Mutations in the N-terminal portion of FADD prevented TNFR1 -induced apoptotic signaling. In addition, the TNFR1-TRADD complex was found to interact with TRAF2 in a pathway required for TNFR1-induced NFxcexaB activation. These findings suggested that TRADD may serve primarily as a linker between TNFR1 and multiple downstream signaling pathways, including FADD-mediated apoptosis. Interestingly, over expression of the death domain of TRADD or RIP alone is sufficient to induce apoptosis, whereas FADD over expression induces apoptosis only if the N-terminal (effector) domain is intact. FADD mutants with altered death domains that are unable to bind Fas are still lethal. Activation of FADD or similar effector proteins may be essential for both TNFR1- and Fas-mediated apoptosis. Recently, FADD has been shown to interact with a novel member of the ICE protease family known as MACH or FLICE. FADD can recruit this protease to the ligand-engaged Fas receptor and promotes the cleavage of the FLICE prodomain, presumably activating the protease. Although such activation may be sufficient to induce apoptosis, Fas may also activate other signaling molecules that contribute to programmed cell death.
Sequential cytological and biochemical changes are associated with the cellular apoptotic process. The cytoplasm condenses, and the endoplasmic reticulum dilates to form vesicles which fuse with the cell membrane, producing characteristic cellular morphology. Changes in the nuclei include the formation of dense crescent shaped aggregates of chromatin, nucleolus fragmentation, and formation of vesicles at or on the nuclear membrane. During apoptosis, endonucleases present in the cell cut the DNA in the linker regions between nucleosomes to release DNA fragments in integer multiples of 180-190 base pairs [J. J. Cohen et al., J. Immunol. 132:38-42 (1984)]. The pattern of cleavage is believed to result from the vulnerability of the linker DNA between the nucleosomes to endonucleases. However, the elucidation of the cellular signaling transduction mechanisms beginning with the apoptosis inducing agent and leading to DNA fragmentation and apoptosis have not been fully determined.
Better understanding and elucidation of various strategic targets in the cellular signaling transduction machinery that leads to DNA fragmentation and apoptosis would provide suitable compounds for modulating cell growth and proliferation by regulation of the apoptotic signaling pathway. What is required are suitable methods and compositions for the identification of compounds that have therapeutic utility where cell growth or proliferation is aberrant, for example, as anti-neoplastic agents.
The present invention generally relates to compositions and methods for identifying apoptosis signaling pathway inhibitors and activators and more particularly, methods and compositions for screening compounds and identifying compounds that will modulate the interactions of the various compositions identified in the present invention; ARC, RICK and the CIDE family of activators (CIDE-A, CIDE-B, DREP-1) with other members of the signaling pathway, i.e., their substrates or ligands. It is not intended that the present invention be limited to particular cell death signaling pathways. The present invention contemplates that the methods and compositions described herein will be useful with a variety of apoptosis-signaling receptors, to identify compounds that will modulate the interactions of ARC, RICK and the CIDE family of activators with other members of the apoptosis signaling pathway, i.e., their substrates or ligands.
In one embodiment, the present invention contemplates an isolated nucleic acid encoding at least a fragment of the RICK protein(RIP-like interacting CLARP kinase), that functions as a positive regulator of apoptosis, having the amino acid sequence (1-531) set forth in SEQ ID NO:1. It is not intended that the present invention be limited by the size or nature of the fragment. In one embodiment, said nucleic acid encodes full-length RICK as set forth in (SEQ ID NO:1) and said nucleic acid comprises SEQ ID NO:2 are contemplated. In another embodiment, the said RICK protein has a mutation at the ATP-binding site, in which the lysine residue is replaced by methionine at position 38 of the amino acid sequence set forth in SEQ ID NO:3. In another embodiment, said nucleic acid encodes a fragment. It is not intended that the present invention be limited by the nature or size of the fragment. For example, fragements comprising either residues 54-531 (SEQ ID NO:4), residues 248-531(SEQ ID NO:5), or residues 365-531 (SEQ ID NO:6), or residues 1-365 (SEQ ID NO:7) of the amino acid sequence set forth in SEQ ID NO:1. In yet another embodiment, said nucleic acid encodes a fusion protein.
It is not intended that the present invention be limited as to the specific nature of the nucleic acid encoding the peptides described above. In one embodiment, said nucleic acid is contained in a vector. In another embodiment, said vector is in a host cell.
In one embodiment, the present invention contemplates screening compounds and identifying compounds that modulate the interactions of the RICK kinase and its substrates, and intracellular proteins in the apoptosis pathway, and in particular CLARP. Furthermore, the present invention contemplates identifying RICK inhibitors and RICK kinase binding substrates, and compounds that will modulate the interaction of RICK kinase with its substrates as well as RICK kinase activity.
In one embodiment, the present invention contemplates identifying compounds that modulate the interaction of RICK kinase, which binds to CLARP. In other embodiments, the present invention contemplates identifying compounds that modulate the interaction of RICK, which binds to FADD.
In preferred embodiments, RICK (and in particular, fragments of RICK as described above) are useful in drug screening assays designed to identify drugs that interfere with the specific binding of RICK kinase with their substrate as well as RICK kinase activity, and thereby block the activation of downstream signaling molecules. In other embodiments, the present invention contemplates identifying compounds that modulate the interaction of RICK, which may bind to substrates other than FADD or proteins other than CLARP.
In general, the present invention contemplates identifying inhibitors that modulate apoptosis mediated by the CD95 pathway, by modulating the interaction of RICK kinase.
In other embodiments, the invention provides an isolated polypeptide, or a fragment thereof, having RICK kinase-specific binding affinity. The invention provides nucleic acids encoding the RICK polypeptide and RICK fragments as part of expression vectors for introduction into cells. The invention provides methods of identifying intracellular molecules which interact with RICK or RICK fragments, as well as exogenous agents (i.e., drugs) which disrupt the binding of RICK and/or fragments thereof to such intracellular targets.
The claimed polypeptide RICK and RICK fragments (see above) find particular use in screening assays for agents or lead compounds for agents useful in the diagnosis, prognosis or treatment of disease, particularly disease associated with undesirable cell growth, and dysregulation of apoptosis.
It is not intended that the present invention be limited by the species (human, murine, rat, etc.) of the binding ligands described above. The polypeptide RICK and RICK fragments shown below may bind across species. Moreover, the nucleic acid sequences described herein allow for the identification of homologues in other species by various methods, including but not limited to amplification (e.g., PCR) using primers designed from the nucleic acid sequence of one species (e.g., human) on the nucleic acid template of another species (e.g., mouse).
In other embodiments, a purified oligonucleotide capable of selectively hybridizing to the nucleic acid molecule set out in FIGS. 7B and 7C (SEQ ID NO:2) is provided, wherein said oligonucleotide comprises a contiguous sequence of at least ten nucleotides (and more preferably at least fifteen nucleotides, and still more preferably at least twenty nucleotides) completely complementary to said nucleic acid molecule set out in FIGS. 7B and 7C (SEQ ID NO:2). In a preferred embodiment, said oligonucleotide is labelled.
The present invention also contemplates complexes of ligands. In one embodiment, the present invention contemplates a composition, comprising a RICK-kinase complex comprising a purified peptide having at least a portion of the amino acid sequence set forth in SEQ ID NO:1 specifically bound to CLARP (or other substrate). Again, the peptides bound specifically to CLARP may be full-length RICK or a fragment defined by a portion of the amino acid sequence as set forth in SEQ ID NO:1. The peptide may be part of a fusion protein. The complex can also contain other ligands, such as FADD and/or the CD95 receptor. The complexes can be used in drug screening assays to identify inhibitor molecules that block CD95-mediated apoptosis (as described below).
As noted above, the present invention contemplates compound screening assays. In one embodiment, the present invention contemplates a method for compound screening, comprising: a) providing: i) a RICK peptide comprising at least a portion of the amino acid sequence set forth in SEQ ID NO:1, wherein said portion is capable of binding to a substrate or protein such as CLARP and/or FADD, ii) RICK substrate, and iii) one or more compounds for screening; b) mixing, in any order, said RICK kinase, said RICK substrate and said one or more compound; and c) measuring the extent of binding of said peptide to said RICK substrate FADD and/or protein CLARP.
One such assay involves forming mixtures of 1) RICK (or fragments thereof) and 2) an intracellular RICK-binding substrate, in the presence or absence of 3) a prospective drug candidate. The mixtures are made under conditions that permit the binding of the intracellular RICK-binding substrate to RICK (or fragments thereof) and the mixtures are then analyzed for the presence of such binding. A difference in such binding in the presence of such a drug candidate indicates that the agent is capable of modulating the binding of RICK (or fragments thereof) to an intracellular RICK-binding substrate.
Another such assay involves forming mixtures of 1) RICK (or fragments thereof) and 2) an intracellular RICK-binding substrate, in the presence or absence of 3) a prospective drug candidate. The mixtures are made under conditions that permit the phosphorylation of the intracellular RICK-binding substrate, such as FADD, by RICK (or fragments thereof) and the mixtures are then analyzed for phosphorylation. A difference in phosphorylation in the presence of such a drug candidate indicates that the agent is capable of modulating the ability of RICK (or fragments thereof) to phosphorylate an intracellular RICK-binding substrate.
Again, the peptides may be full-length RICK or a fragment defined by a portion of the amino acid sequence as set forth in SEQ ID NO:1. The peptide may also be part of a fusion protein. The present invention also contemplates embodiments where either the peptide or kinase is bound to other ligands. These complexes can be used in the compound screening assay described above.
In another preferred embodiment, the present invention contemplates an isolated nucleic acid encoding at least a fragment of the Apoptosis Repressor Protein, ARC having the amino acid sequence (1-208) set forth in SEQ ID NO:8. It is not intended that the present invention be limited by the size or nature of the fragment. In one embodiment, said nucleic acid encodes full-length ARC as set forth in (SEQ ID NO:8) and said nucleic acid comprises SEQ ID NO:9. In another embodiment, said nucleic acid encodes a fragment comprising either residues 1-106 (SEQ ID NO:10) or residues 107-208 (SEQ ID NO:11) of the amino acid sequence set forth in SEQ ID NO:8. In yet another embodiment, said nucleic acid encodes a fusion protein.
In other embodiments, a purified oligonucleotide capable of selectively hybridizing to the nucleic acid molecule set out in FIG. 1D (SEQ ID NO:9) is provided, wherein said oligonucleotide comprises a contiguous sequence of at least ten nucleotides (and more preferably at least fifteen nucleotides, and still more preferably at least twenty nucleotides) completely complementary to said nucleic acid molecule set out in FIG. 1D(SEQ ID NO:9). In a preferred embodiment, said oligonucleotide is labelled.
It is not intended that the present invention be limited as to the specific nature of the nucleic acid encoding the peptides described above. In one embodiment, said nucleic acid is contained in a vector. In another embodiment, said vector is in a host cell.
In preferred embodiments, over expression of ARC or fragments thereof, in an in vitro cell system can be used for identifying similar inhibitors that inhibit the enzymatic activity of caspase-8 and thereby block CD95-mediated apoptosis. Also, identification of ARC-like inhibitory compounds may be particularly useful for gene therapy, for e.g., for the treatment of diseases that are characterized by inappropriately increased cell death in muscle tissue and cardiac disorders.
In other preferred embodiments, the present invention contemplates an isolated nucleic acid encoding at least a fragment of the Human CIDE-A protein (Cell Death inducing DFF45-like effector-A) having the amino acid sequence (1-219) set forth in SEQ ID NO:12. It is not intended that the present invention be limited by the size or nature of the fragment. In one embodiment, said nucleic acid encodes full-length CIDE-A as set forth in (SEQ ID NO:12) and said nucleic acid comprises SEQ ID NO:13. In another embodiment, said nucleic acid encodes a fragment comprising either residues 1-107 (SEQ ID NO:14), or residues 108-200 (SEQ ID NO:15) of the amino acid sequence set forth in SEQ ID NO:12. In yet another embodiment, said nucleic acid encodes a fusion protein.
In other embodiments, a purified oligonucleotide capable of selectively hybridizing to the nucleic acid molecule set out in FIG. 14E (SEQ ID NO:13) is provided, wherein said oligonucleotide comprises a contiguous sequence of at least ten nucleotides (and more preferably at least fifteen nucleotides, and still more preferably at least twenty nucleotides) completely complementary to said nucleic acid molecule set out in FIG. 14E (SEQ ID NO:13). In a preferred embodiment, said oligonucleotide is labelled.
In other preferred embodiments, the present invention contemplates an isolated nucleic acid encoding at least a fragment of the Mouse CIDE-A protein (Cell Death inducing DFF45-like effector-A) having the amino acid sequence (1-217) set forth in SEQ ID NO:16. It is not intended that the present invention be limited by the size or nature of the fragment. In one embodiment, said nucleic acid encodes full-length CIDE-A as set forth in (SEQ ID NO:16) and said nucleic acid comprises SEQ ID NO:17.
In yet other embodiments, a purified oligonucleotide capable of selectively hybridizing to the nucleic acid molecule set out in FIG. 14G (SEQ ID NO:17) is provided, wherein said oligonucleotide comprises a contiguous sequence of at least ten nucleotides (and more preferably at least fifteen nucleotides, and still more preferably at least twenty nucleotides) completely complementary to said nucleic acid molecule set out in FIG. 4G (SEQ ID NO:17). In a preferred embodiment, said oligonucleotide is labelled.
In yet other preferred embodiments, the present invention contemplates an isolated nucleic acid encoding at least a fragment of the Mouse CIDE-B protein (Cell Death inducing DFF45-like effector-B) having the amino acid sequence (1-219) set forth in SEQ ID NO:1 8. It is not intended that the present invention be limited by the size or nature of the fragment. In one embodiment, said nucleic acid encodes full-length CIDE-B as set forth in (SEQ ID NO:18) and said nucleic acid comprises SEQ ID NO:19. In yet another embodiment, said nucleic acid encodes a fusion protein.
In yet other embodiments, a purified oligonucleotide capable of selectively hybridizing to the nucleic acid molecule set out in FIG. 14I (SEQ ID NO:19) is provided, wherein said oligonucleotide comprises a contiguous sequence of at least ten nucleotides (and more preferably at least fifteen nucleotides, and still more preferably at least twenty nucleotides) completely complementary to said nucleic acid molecule set out in FIG. 14I (SEQ ID NO:19). In a preferred embodiment, said oligonucleotide is labelled.
In other preferred embodiments, the present invention contemplates an isolated nucleic acid encoding at least a fragment of the Drosophila DREP-1 protein (DFF45-homologue) having the amino acid sequence (1-299) set forth in SEQ ID NO:20. In one embodiment, said nucleic acid encodes full-length DREP-1 as set forth in (SEQ ID NO:20) and said nucleic acid comprises SEQ ID NO:21. It is not intended that the present invention be limited by the size or nature of the fragment. In one embodiment, said nucleic acid encodes a fusion protein.
In yet other embodiments, a purified oligonucleotide capable of selectively hybridizing to the nucleic acid molecule set out in FIG. 14K (SEQ ID NO:21) is provided, wherein said oligonucleotide comprises a contiguous sequence of at least ten nucleotides (and more preferably at least fifteen nucleotides, and still more preferably at least twenty nucleotides) completely complementary to said nucleic acid molecule set out in FIG. 14K (SEQ ID NO:21). In a preferred embodiment, said oligonucleotide is labelled.
It is not intended that the present invention be limited as to the specific nature of the nucleic acid encoding the various peptide compositions described above. In one embodiment, said nucleic acid is contained in a vector. In another embodiment, said vector is in a host cell.
In preferred embodiments, therapeutic compositions of CIDEs can be used for the treatment of diseases and/or cancer, as DFF45-inhibitable effectors that promote